This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glycosphingolipids (GSLs) and phospholipids (PLs) are components of cell membranes that regulate their biophysical properties and participate in diverse biological processes. Human milk sulfatides, but not those from bovine brain, are active against HIV infection of human macrophages and lymphocytes. The biological roles are dependent on the specific oligosaccharide and the ceramide portions. The polar headgroup of PLs may consist of ethanolamine, choline, serine, inositol, or phosphatidylglycerol. The acyl groups may differ in their degrees of saturation/ hydroxylation and in their fatty acyl chains. We are using vibrationally cooled MALDI-FTMS for the detection of TLC-separated species followed by their efficient fragmentation by SORI-CAD and IRMPD to determine structural details. Lipid standards or lipids extracted from human milk were separated by TLC using chloroform/methanol/acetic acid/water (65;25;3;1 v/v);the plate was then sprayed with the saturated matrix solution, affixed to the target, and scanned by VC-MALDI-FTMS. Fragmentation was performed by SORI-CAD with N2 collision gas and IRMPD techniques. The lipids were MALDI-desorbed directly off TLC plate surfaces and thermalized by the cooling gas. The peaks of interest were isolated by SWIFT and fragmented by SORI-CAD and IRMPD. Numerous neutral and acidic GSL and PL homologs were found following each scanning step. Short IRMPD pulses defined the glycan moiety and higher energy irradiation established the ceramide structures. Application of SORI-CAD and IRMPD to the sulfatides resulted in high-abundance peaks characteristic of HSO4- and hexose sulfate. Fatty acyl chain lengths varied (C16 - C24);some were hydroxylated. Four major species desorbed from a single TLC spot produced (+) mode spectra showing sequential elimination of two oleoyl groups;(-) mode MS obtained from these spots showed evidence for the loss of C16:0, C18:0, C18:1 and C18:2 acyl chains. Analysis of two spots with lower Rf values by SORI-CAD indicated the presence of a glycerophospholipid with palmitoyl and oleoyl (34:1) or palmitoyl and linoleoyl (34:2) substituents, and two spots at high Rf had linoleoyl and stearoyl (36:2) and linoleoyl and oleoyl (36:3) substituents. Other unusual phosphatidyl derivatives were also present and are being further investigated. Current studies also employ MALDI-TOF MS on the Bruker Reflex IV and LC/MS/MS on the Q-Star and LTQ-Orbitrap MS to assign glycolipid structures.